Transcript New TURF for TIRF - Stowers Institute for Medical Research

New TURF for TIRF
Joel Schwartz
Stowers Institute for Medical Research
Imaging Center
• What is TIRF?
• Why do we constantly use acronyms to
describe everything?
• Microscope Configurations
– Prism vs Prismless
• Biological Applications
– Brief Aside
• Unique attributes to our system
– Calibrated TIRF planes
– TIRF-FRET
– TIRF-photoactivation
• Not ready for prime time players..
• What is TIRF?
• Why do we constantly use acronyms to
describe everything?
• Microscope Configurations
– Prism vs Prismless
• Biological Applications
– Brief Aside
• Unique attributes to our system
– Calibrated TIRF planes
– TIRF-FRET
– TIRF-photoactivation
• Not ready for prime time players..
Index of refraction “bends” light
Some refractive indices to know:
water
1.33
air
1.0003
glass
1.517
coverglass
1.523
immersion oil
1.516
cell cytosol
1.38
mount
variable
The basics of imaging cells by TIRF microscopy
At a specific critical angle [θcritical = sin-1(n1/n2)] light is totally reflected from the glass/water interface. The
reflection generates a very thin electromagnetic field that has an identical frequency to that of the incident
light, providing a means to selectively excite fluorophores within ≤ 100 nm of the coverslip.
http://micro.magnet.fsu.edu/primer/java/tirf/penetration/index.html
Evanescent wave penetration
The evanescent wave penetration (d) :
d = λ0/4π (n22 sin2θ-n12)-1/2
note: d is only the depth at which the intensity of the evanescent wave is 37% of the initial
intensity. Thus, can empirically determine the experimental depth at which fluorophores are
visible using fluorescent beads (Keyel, Watkins, and Traub 2004 JBC)
λ0 = 488; n2=1.52; n1=1.38 dempirical = 190 nm
λ0 = 647; n2=1.52; n1=1.38 dempirical = 238 nm
λ0 = 488; n2=1.78; n1=1.38 dempirical = 142 nm
TIRF selectively illuminates the
cellular membrane
• What is TIRF?
• Why do we constantly use acronyms to
describe everything?
• Microscope Configurations
– Prism vs Prismless
• Biological Applications
– Brief Aside
• Unique attributes to our system
– Calibrated TIRF planes
– TIRF-FRET
– TIRF-photoactivation
• Not ready for prime time players..
Prism-based TIRF limit access to sample
Axelrod et al. Traffic 2001
Prism systems can be placed under a culture dish
Prism-based TIR on an upright microscope
Trapezoid TIR prism on condenser and the position of the beam is
adjusted by moving external lens.
Axelrod et al. Traffic 2001
TIRF is commonly done inside the
objective
The objective influences penetration depth
100X 1.45 NA objective:
θc = sin-1(n1/n2) = 65.22º
[calculated using n2 = 1.52 (RI coverglass and immersion liquid) and n 1 = 1.38]
100X 1.65 NA objective:
θc = sin -1(n1/n2) = 50.83º
[calculated using n2 = 1.78 (RI coverglass and immersion liquid) and n 1 = 1.38]
Maximum Angle θm from the optical axis that TIR will occur is:
NA = n2 sin θm
60X 1.45 NA θm = 72.54º
100X 1.65 NA θm = 67.97º
TIRF objectives are now starting to come with compensation collars for varying
temperature and cover slip thickness
TIRF Comparison
Prism Method
1. “Purer” evanescent wave
2. Limited access to sample
3. Few commercial manufactures
4. Open laser systems
5. Typically lower NA objectives
Prism-less Method
1. Higher NA will allow confinement closer to surface
2. Not as pure an evanescent wave as prism
3. Commercial system readily available
• What is TIRF?
• Why do we constantly use acronyms to
describe everything?
• Microscope Configurations
– Prism vs Prismless
• Biological Applications
– Brief Aside
• Unique attributes to our system
– Calibrated TIRF planes
– TIRF-FRET
– TIRF-photoactivation
• Not ready for prime time players..
TIRF illumination enhances contrast
YFP on the Membrane
TIRF is more sensitive to Z-axial drift
Hogan, Biophotnics International May 2006 48-51
TIRF measures endocytosis of clathrin
coated vesicles
Color Coded Motion
Red
Green
Blue
RGB
Clathrin coated pits are move in and
out of the membrane
Membrane-localized fluorophores are difficult to
separate from mitochondria
TIRF selectively visualizes the membrane localized
fluorophores
Total Internal Reflection Fluorescence (TIRF)
Microscopy is used to reduce background
Applications of TIR microscopy
•Selective visualization of cell/substrate contact regions.
•Visualization and spectroscopy of single molecule fluorescence near a
surface.
•Tracking of secretory granules in intact cells before and during the
secretory process.
•Micromorphological structures and dynamics on living cells.
•Long-term fluorescence movies of cells during development in culture.
•Comparison of membrane-proximal ionic transients with simultaneous
transients deeper in the cytoplasm.
•Measurements of the kinetic rates of binding of extracellular and
intracellular proteins to cell surface receptors and artificial membranes.
• What is TIRF?
• Why do we constantly use acronyms to
describe everything?
• Microscope Configurations
– Prism vs Prismless
• Biological Applications
– Brief Aside
• Unique attributes to our system
– Calibrated TIRF planes
– TIRF-FRET
– TIRF-photoactivation
• Not ready for prime time players..
The new rig
405, 440, 491, 561, 638
AOTF operated
Axiocam HS
Back-thinned
EM-CCD
BAD IDEA!
Environmental Chamber
High tech TIRF calibration device
Fluorescencce (Epi)
1.0
0.8
Red =120nm
Green=88nm
0.6
0.4
0.2
0.0
0
100
200
300
Distance (nm)
400
500
FRET measures protein proximity
TIRF enhances signal to noise measurements of
membrane associated FRET
1 um z-axial
~ 15 receptors
~ 75 associated proteins
100 nm
~ 15 receptors
~ 15 associated proteins
We idealized the system to excite CFP
for FRET measurements
Excitation of CFP leads to some YFP excitation because YFP is ~5
fold brighter than CFP. CFP emission also bleeds into the YFP
channel (i.e. there will always be some “FRET” signal).
The new TIRF scope is capable of specific
membrane photoactivation
• What is TIRF?
• Why do we constantly use acronyms to
describe everything?
• Microscope Configurations
– Prism vs Prismless
• Biological Applications
– Brief Aside
• Unique attributes to our system
– Calibrated TIRF planes
– TIRF-FRET
– TIRF-photoactivation
• Not ready for prime time players..
Spectral images separate overlaping spectra
Dickinson et al. Biotechniques. 31:1272
2001.
CTIS provides space and color information
without any moving parts
NOT YET AVAILABLE
The system is a linear transfer function similar to CT
scanning and reconstruction
Spectral Imaging: CTIS
Space and Color in a Single Shot
CGH Disperser
The CTIS images are deconvolved to generate
the actual image
Ford et al., Optics Express’01 (9) 444-453.
Raw Data on CCD
Color projection of final data stack
Thank You
• Imaging Center
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Cameron Cooper
Paul Kulesa
Sarah Smith
Danny Stark
Jessica Teddy
Miranda Smith
• Adv. Inst. And Physics
– Winfried Wiegraebe
– Josef Huff
– Amanda Combs
http://micro.magnet.fsu.edu/primer/java/tirf/evaintensity/index.html